The invention relates generally to methods for detecting an alteration in a target nucleic acid.
Many diseases are associated with genomic instability. As such, instability markers have been proposed as diagnostics. For example, mutations are considered valuable markers for a variety of diseases, and have formed the basis for screening assays. Specific mutations might be a basis for molecular screening assays for the early stages of certain types of cancer. See, e.g., Sidransky, et al., Science, 256: 102-105 (1992). For example, mutations in the BRCA genes have been proposed as markers for breast cancer, and mutations in the p53 cell cycle regulator gene have been associated with the development of numerous types of cancers.
Early alteration detection allows early disease diagnosis, and thus also provides an avenue for intervention prior to the presentation of disease symptoms that often occurs after metastasis when a cure is less readily attainable. However, the detection of genetic mutations or other alterations is difficult, or impossible, in certain sample types. For example, the difficulty of isolating DNA from complex, heterogeneous samples makes identification of early-stage mutation difficult.
Therefore, there is a need in the art for efficient methods for determining the presence or absence of certain genetic mutations or other alterations in a target nucleic acid in a biological sample.
The invention provides methods for detecting an alteration in a target nucleic acid in a biological sample. According to the invention, a series of nucleic acid probes complementary to a contiguous region of wild type target DNA are exposed to a sample suspected to contain the target. Probes are designed to hybridize to the target in a contiguous manner to form a duplex comprising the target and the contiguous probes xe2x80x9ctiledxe2x80x9d along the target. An example of this duplex is shown in FIG. 1. If a mutation or other alteration exists in the target, contiguous tiling will be interrupted, producing regions of single-stranded target in which no duplex exists. This is shown in FIG. 2. Identification of one or more single-stranded regions in the target is indicative of a mutation or other alteration in the target that prevented probe hybridization in that region. For purposes of the present invention, a xe2x80x9ctiled sequencexe2x80x9d or xe2x80x9ctilingxe2x80x9d refers to the contiguous hybridization of probes to a target region, whether separated by single-stranded sequence or not.
Accordingly, in methods of the invention, a sample comprising a single-stranded target nucleic acid is exposed to a plurality of nucleic acid probes. In a preferred embodiment, the plurality of probes comprises probes that are complementary to different positions of the target such that hybridization of members of the plurality with a wild-type target results in a contiguous series of probes along at least a portion of the target sequence when the target is a wild-type target. Probes including RNA, DNA and/or Peptide Nucleic Acid (PNA) may be employed to hybridize to the target nucleic acid. It is not necessary to ligate the series of probes to form a continuous strand, although ligation may be performed at the discretion of the user.
When the target is a wild-type sequence, there will be no single-stranded portion in the region in which the probes are tiled. However, when a mutation or other alteration exists in the region of the target to which probes are directed, one or more of the probes will fail to hybridize, resulting in one or more single-stranded portion of the target region. Identification of this single-stranded region is, according to the invention, a positive assay for a mutation or other alteration in the target.
In a preferred embodiment, a single-stranded region indicative of a mutation in the target is detected by exposing the target, subsequent to probe hybridization, to an agent that selectively cleaves single-stranded nucleic acid. In a mutated target, methods of the invention produce more than one xe2x80x9ctiledxe2x80x9d duplex in the target region. Multiple double-stranded tiled duplexes result from cleavage of the target in the single-stranded region to which any probe failed to hybridize. Numerous cleavage enzymes are known which selectively cleave or degrade single-stranded nucleic acids (e.g., S1, MutY, MutS and Mungbean nuclease). Identification of a single contiguous duplex comprising the target and the contiguous tiled probes upon exposure to the selective cleavage or degradation agent is indicative of a wild-type (non-mutated) target region. Alternatively, the products of cleavage are measured to determine, for example, whether the molecular weight of the products is different than would be expected from a single contiguous duplex.
Also in a preferred embodiment, the assay described above is multiplexed in order to interrogate multiple targets simultaneously. As such, one can look for specific double-stranded cleavage products in order to identify the specific mutated target(s) or one can simply identify multiple cleavage products (resulting, as described above, from intervening single-stranded regions in the xe2x80x9ctiled targetxe2x80x9d) as evidence of a mutation at one of the interrogated targets. For example, multiple targets, each containing a so-called xe2x80x9chot spotxe2x80x9d for mutation in cancer are interrogated, the production of a single-stranded target region after tiling being sufficient to result in a positive screen for cancer or pre-cancer.
Methods of the invention are also useful for detecting non-hybridized regions at the termini of a target. When a mutation occurs in a region of target to which a terminal tile would hybridize if the target is a wild-type target, the resulting degradation of the single-stranded terminus will not, as described above, produce multiple duplex products indicative of an intervening single-stranded region. Instead, the terminal single-stranded region will be cleaved or degraded, leaving the tiled portion of the target intact. In that case, the terminal mutation is identified in by the reduced expected molecular weight of the tiled target or by the activity of the degrading agent (e.g., an exonuclease).
Alternatively, a mutation or other alteration in the termini of a target may also be detected by evaluating both the sense strand and antisense strand of the target. According to methods of the invention, both the sense and antisense strands of the target are bound to a solid support by the same respective terminus; for example, both the sense and the antisense strands of the target are bound to a solid support by their respective 5xe2x80x2 ends. Thereafter, the bound sense and antisense strands of the target are interrogated in solution. A terminal mutation on, for example, the unbound 3xe2x80x2 end of the sense strand would go undetected, however, the mutation presents a duplex cleaved from the mutation site near the bound 5xe2x80x2 end of the antisense strand. The mutation is detected when the solid support is removed and the duplex cleaved off of the antisense strand remains in solution. If only the sense strand were tested, then the mutation would go undetected, thus testing both the sense and the antisense strands avoids a false negative caused by a terminal mutation on one of the strands.
In another embodiment, the invention provides methods to avoid detecting a known polymorphism as a false positive for an alteration. One or more polymorphisms may be present in a region of the target nucleic acid. In that case, multiple probes are designed to hybridize to each of the polymorphic variants known to be present in the target region. The probe complimentary to the polymorphism variant that is present in the target will hybridize to the region of the polymorphism. Providing the range of variants complementary to the associated polymorphisms ensures that the polymorphism region is xe2x80x9ctiledxe2x80x9d and any positive signal detected indicates the presence of an alteration other than the associated polymorphism on the target nucleic acid.
Methods of the invention are also useful for detecting the presence, in a population, of one or more polymophisms on a target nucleic acid. A plurality of nucleic acid probes is complementary to a first variant of a target nucleic acid comprising a polymorphism such that hybridization of members of the plurality with the target comprising the polymorphism variant results in a contiguous series of probes along at least a portion of the target. When one or more other polymorphic variants are present in the region of the target to which the probes are directed, at least one of the probes will fail to hybridize at the site of the other polymorphic variant. After hybridization, a single-stranded portion of the target region is exposed at the site of, for example, a second polymorphic variant. Upon exposure to an agent that selectively degrades single-stranded nucleic acid, the polymorphic variant presents a duplex cleaved from, for example, the site of the second polymorphic variant. Accordingly, detection of a cleavage product is a positive assay for an alteration, in this case the one or more other polymorphic variants, in the target.
In another aspect, probes are designed to hybridize to the target such that gaps separate one or more of the hybridized probes from each other. According to this aspect of the invention, a sample comprising single-stranded target nucleic acid is exposed to a plurality of nucleic acid probes. The plurality comprises probes that are complementary to different positions on the target nucleic acid such that hybridization of members of the plurality with a wild-type target results in a series of probes, along at least a portion of the target sequence, some of which may be separated a gap. Preferably, the gaps are small enough to prevent the degradation agent from cutting at the gap. Accordingly, preferred gap separations are shorter than the probes, i.e., the probes have a higher number of base pairs then the gaps. In preferred embodiments, the gap separations range between about 1 base pair and about 3 base pairs in length. Alternatively, gaps can be between about 3 base pairs and 15 base pairs in length. According to the invention, a detection assay can be performed using a plurality of probes some of which are separated by gaps and some of which are not when hybridized to the target nucleic acid.
According to this aspect of the invention, the degradation agent that that is selected, or alternatively, the conditions employed with the agent, do not cleave the target nucleic acid at the single-stranded gaps. In some embodiments, milder degradation conditions that avoid degrading any single-stranded gap separations are employed. For example, the number of degradation agent units used are held at a temperature and for a time period appropriate to maintain the single-stranded gap separations between tiles. The degradation agent conditions simultaneously degrade larger single-stranded regions where one or more probes failed to hybridize due to an alteration in the target. Because the gap separations are not degraded, false positives created by gap separations between the probes are avoided. However, the conditions enable degradation of the target region where the probe failed to hybridize, avoiding a false negative. Thus, according to the invention, a positive assay for an alteration in a target nucleic acid may comprise gaps separating complementary probes hybridized to the target. Where gap separations are present between probes, the selected degradation agent, units of agent used, temperature and exposure time are selected to provide conditions that maintain single stranded gap separations and that simultaneously cleave a single stranded alteration region where a probe failed to hybridize. Steric hindrance can also be used to prevent cutting at the gaps.
In another embodiment of the invention, the probes may be designed such that they prevent adjacent hybridized probes from creating a stabilizing effect that results in a probe hybridizing at the site of an alteration in a region of the target nucleic acid. Accordingly, the probes themselves are altered to include between about 1 and about 10 Abasic sites on the 5xe2x80x2 and/or the 3xe2x80x2 end of one or more of the probes. Similarly, the probes may be altered to put between about 1 to about 10 DNA, RNA, or Peptide Nucleic Acids that do not match the target nucleic acid on the 5xe2x80x2 and/or the 3xe2x80x2 end, and that do not prevent the probe from hybridizing to its complementary target.
In a preferred embodiment, a target nucleic acid is bound to a solid-support at either its 3xe2x80x2 or 5xe2x80x2 terminus. Complementary probes are tiled along the length of the target as described above. A mutation is indicated when double-stranded hybridization products are detected in solution after the sample is treated with a degradation agent indicating that one or more tiling probes failed to hybridize to the target due to the mutation. More than one target nucleic acid from more than one source can be simultaneously screened by binding multiple target nucleic acids to solid supports. Also, double-stranded nucleic acid according to the invention can be melted by, for example, heating.
In the event that a mutation is detected on a target nucleic acid, the identity of the mutation is determined by any method known in the art, such as sequencing, mass spectroscopy, and others.
In a preferred embodiment, a biological sample is exposed to probes complementary to a target DNA under stringent hybridization conditions so that each probe will hybridize only to the wild-type target nucleic acid. Such conditions are well-known in the art. See, e.g., 2 Joseph Sambrook, Peter MacCallum, and David Russell, Molecular Cloning: A Laboratory Manual ch., 10 (3d ed. 2001), incorporated by reference herein. In one embodiment, the hybridization melting temperature of each probe is about the same. In another embodiment, the probes are between about 8 and about 30 nucleotides long. In one preferred embodiment, each probe is the same length i.e. composed of the same number of nucleotides.
Preferred biological samples are sputum, pancreatic fluid, bile, lymph, plasma, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, pus, biopsy tissue, fetal cells, amniotic fluid, and stool.
In another embodiment, at least one of the tiling probes comprises a detectable label. Each probe may comprise a different detectable label, permitting the differential detection of the probes (i.e., for example, the different probes may comprise a nucleotide with a different radioactive isotope, a fluorescent tag, or a molecular weight modifying entity). Differential probe labeling allows the identification of the probe that did not anneal to its target in the case of a mutation.
In another embodiment, the target nucleic acid comprises a detectable label in the region at which a mutation is suspected. When the suspected mutation is present in the target, no probe will hybridize to the target and the region of the mutation comprising the detectable label will remain single stranded. Upon exposure to an agent that cleaves single-stranded nucleic acid, the single-stranded mutation region comprising the detectable label is degraded from the target. The absence of the label in the degradation products is indicative of the presence of a mutation in the region of the detectable label.
In another embodiment, a quencher is placed at one end and a reporter is placed at the other end of each of the probes such that the absence of hybridization of one probe results in the reporter of an adjacent probe failing to be quenched. Accordingly, an alteration is detected by the reporter signal. The reporter may be detected via a fluorescent plate reader or alternatively with a gel apparatus equipped with fluorescent detection.
In yet another embodiment, a catalyst is placed at one end and an inhibitor is placed at the other end of each of the probes such that the absence of hybridization of one probe results in the exposure an uninhibited catalyst. Accordingly, an alteration is detected by a signal (e.g., fluorescence) generated by addition of an enzyme catalyzed by the uninhibited catalyst.
In one embodiment, methods of the invention comprise detecting a mutation at a genetic locus that is associated with a disease, such as K-RAS, p53, APC, DCC, or BAT26. In a preferred embodiment, that mutation is associated with cancer, such as colon cancer, lung cancer, esophageal cancer, prostate cancer, breast cancer, pancreatic cancer, stomach cancer, liver cancer, or lymphoma.